To investigate whether the cell dispersion techniques commonly employed to harvest monolayer cultures of tumor cells for injection into experimental animals might induce alterations in cellular biochemistry, we compared the phosphoprotein profiles of 6 B16 melanoma clones of distinct metastatic potential using 2-D gel electrophoresis after growth in monolayer culture, after suspension by treatment, with trypsin/EDTA and after injection of suspended cells into syngeneic mouse plasma. Trypsin/EDTA treatment and subsequent exposure to syngeneic mouse plasma induced significant alterations in phosphoprotein composition in all clones. Most alterations were quantitative, involving either enhanced or diminished expression of specific phosphoproteins, but qualitative changes involving expression of novel phosphoproteins were also observed. None of the changes in phosphoprotein composition correlated with metastatic potential. The principle alteration induced in all clones by trypsin/EDTA involved enhanced phosphorylation of an NP-40-soluble component with a molecular weight of 79,000 and an isoelectric point of 6.3 [pp79 (6.3)]. This determinant was detected in extracts of B16 monolayer cultures but its level of phosphorylation was enhanced significantly by trypsin/EDTA treatment and by exposure of the harvested cells to syngeneic mouse plasma. These data indicate that procedures commonly employed to harvest tumor cells for assay of tumorigenic and metastatic potential may provide extensive alterations in phosphoprotein composition and that biochemical investigations of tumor cells grown in monolayer culture may not accurately reflect the metabolic status of the same cells immediately prior to and following i.v. injection into experimental animals.